Modified AXL Peptides and Their Use in Inhibition of AXL Signaling in Anti-Metastatic Therapy

ABSTRACT

Compositions and methods are provided for alleviating cancer in a mammal by administering a therapeutic dose of a pharmaceutical composition that inhibits activity of AXL, MER or Tyro3 protein activity, for example by competitive or non-competitive inhibition of the binding interaction between AXL, MER or Tyro3 and its ligand GAS6.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 15/783,850, filed Oct. 13, 2017, which is a Continuation ofapplication Ser. No. 14/650,854 filed Jun. 9, 2015, now U.S. Pat. No.9,822,347,issue Nov. 21, 2017, which is a 371 application and claims thebenefit of PCT Application No. PCT/US2013/074786, filed Dec. 12, 2013,which claims benefit of U.S. Provisional Patent Application No.61/737,276, filed Dec. 14, 2012, which applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to tumor invasion and metastasis, e.g.,treatments or diagnoses of tumor invasion or metastasis via pathwaysrelated to AXL, MER and Tyro3 and/or GAS6.

BACKGROUND OF THE INVENTION

Invasion and metastasis are the most insidious and life-threateningaspects of cancer. While tumors with minimal or no invasion may besuccessfully removed, once the neoplasm becomes invasive, it candisseminate via the lymphatics and/or vascular channels to multiplesites, and complete removal becomes very difficult. Invasion andmetastases kill hosts through two processes: local invasion and distantorgan colonization and injury. Local invasion can compromise thefunction of involved tissues by local compression, local destruction, orprevention of normal organ function. The most significant turning pointin cancer, however, is the establishment of distant metastasis. Thepatient can no longer be cured by local therapy alone at this point.

The process of metastasis is a cascade of linked sequential stepsinvolving multiple host-tumor interactions. This complex processrequires the cells to enter into the vascular or lymphatic circulation,arrest at a distant vascular or lymphatic bed, actively extravasate intothe organ interstitium and parenchyma, and proliferate as a secondarycolony. Metastatic potential is influenced by the localmicroenvironment, angiogenesis, stroma-tumor interactions, elaborationof cytokines by the local tissue, and by the molecular phenotype of thetumor and host cells.

Local microinvasion can occur early, even though distant disseminationmay not be evident or may not yet have begun. Tumor cells penetrate theepithelial basement membrane and enter the underlying interstitialstroma during the transition from in situ to invasive carcinoma. Oncethe tumor cells invade the underlying stroma, they gain access to thelymphatics and blood vessels for distant dissemination while releasingmatrix fragments and growth factors. General and widespread changesoccur in the organization, distribution, and quantity of the epithelialbasement membrane during the transition from benign to invasivecarcinoma.

Therapeutic efforts in cancer prevention and treatment are being focusedat the level of signaling pathways or selective modulatory proteins.Protein kinase activities, calcium homeostasis, and oncoproteinactivation are driving signals and therefore may be key regulatory sitesfor therapeutic intervention. Kinases in signaling pathways regulatinginvasion and angiogenesis may be important regulators of metastasis. Oneof the largest classes of biochemical molecular targets is the family ofreceptor tyrosine kinases (RTKs). The most common receptor tyrosinekinase molecular targets to date are the EGF and vascular endothelialgrowth factor (VEGF) receptors. Newer kinase molecular targets includethe type III RTK family of c-kit, and abl. Inhibitors of these moleculeshave been administered in combination with classic chemotherapeutics.

Metastases ultimately are responsible for much of the suffering andmortality from cancer. A need exists to identify and target molecularand functional markers that identify metastatic cancer cells and togenerate reagents for their specific inhibition.

Publications in this field include, inter alia, Li et al. Oncogene.(2009) 28(39):3442-55; United States Patent Application, 20050186571 byUllrich et al.; United States Patent Application 20080293733 by Bearsset al.; Sun et al. Oncology. 2004; 66(6):450-7; Gustafsson et al. ClinCancer Res. (2009) 15(14):4742-9; Wimmel et al. Eur J Cancer. 200137(17):2264-74; Koorstra et al. Cancer Biol Ther. 2009 8(7):618-26; Taiet al. Oncogene. (2008) 27(29):4044-55

The receptor tyrosine kinase AXL (also known as Ufo and Tyro7) belongsto a family of tyrosine receptors that includes Tyro3 (Sky) and Mer(Tyro12). A common ligand for AXL family is GAS6 (Growth arrest-specificprotein 6). Human AXL is a 2,682-bp open reading frame capable ofdirecting the synthesis of an 894-amino acid polypeptide. Two variantmRNAs have been characterized, transcript variant 1 may be accessed atGenbank, NM_021913.3 and transcript variant 2 may be accessed atNM_001699.4. The polypeptide sequence of the native protein is providedas SEQ ID NO:1, and specific reference may be made to the sequence withrespect to amino acid modifications. Important cellular functions ofGAS6/AXL include cell adhesion, migration, phagocytosis, and inhibitionof apoptosis. GAS6 and AXL family receptors are highly regulated in atissue and disease specific manner.

AXL, MER and Tyro3 are each characterized by a unique molecularstructure, in that the intracellular region has the typical structure ofa receptor tyrosine kinase and the extracellular domain containsfibronectin III and Ig motifs similar to cadherin-type adhesionmolecules. During development, AXL, MER and Tyro3 are expressed invarious organs, including the brain, suggesting that this RTK isinvolved in mesenchymal and neural development. In the adult, AXL, MERand Tyro3 expression is low but returns to high expression levels in avariety of tumors. GAS6 is, so far, the single, activating ligand forAXL, MER and Tyro3.

Receptor tyrosine kinases (RTK) are generally activated by ligands thatpromote receptor dimerization and, in turn, autophosphorylation oftyrosine residues within the cytosolic domain. Binding of signalingproteins to these phosphorylated tyrosine residues then leads todownstream signaling. AXL, MER and Tyro3 family of RTKs are unique inthat they are activated by GAS6, members of the vitamin K-dependentprotein family that resembles blood coagulation factors rather thantypical growth factors.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that AXL, MERand Tyro3 and/or GAS6 related pathways are related to tumor invasionand/or metastasis. Accordingly, the present invention providescompositions and methods useful for treating tumor invasion and/ormetastasis, e.g., via inhibition of AXL, MER and/or Tyro3 and/or GAS6related pathways. In addition, the present invention provides reagentsand methods useful for determining the susceptibility or likelihood of atumor to become invasive and/or metastatic, e.g., via detecting thelevel of activity of AXL, MER, Tyro3 and/or GAS6.

In some embodiments, the agent useful for treating tumor invasion and/ormetastasis, e.g., via inhibition of AXL, MER and Tyro3 and/or GAS6related pathways is an inhibitor agent. In some embodiments, theinhibitor agent is selected from the group consisting of (a) aninhibitor of AXL, MER and/or Tyro3 activity, (b) an inhibitor of GAS6activity and (c) and inhibitor of AXL, MER and/or Tyro3-GAS6interaction, wherein the inhibitor agent is capable of binding to GAS6with increased affinity compared to wild-type AXL, MER or Tyro3.

In some embodiments, the inhibitor agent binds to two or more epitopeson a single GAS6.

In some embodiments, at least one of the epitopes is the major or minorAXL, MER or Tyro3 binding site on GAS6.

In some embodiments, the inhibitor agent is capable of binding to themajor and minor AXL, MER or Tyro3 binding sites on a single GAS6.

In some embodiments, the inhibitor agent is capable of binding to themajor AXL, MER or Tyro3 binding site of GAS6 and one or more additionalGAS6 epitopes on a single GAS6.

In some embodiments, the inhibitor agent is capable of binding to theminor AXL, MER or Tyro3 binding site on GAS6 and one or more additionalepitopes on a single GAS6.

In some embodiments, the inhibitor agent is capable of binding two ormore epitopes on a single GAS6.

In some embodiments, the inhibitor agent is capable of antagonizing themajor and/or minor GAS6/receptor binding interaction, where the receptoris selected from AXL, MER and Tyro3.

In some embodiments, the inhibitor agent is capable of antagonizing themajor GAS6/receptor binding interaction, where the receptor is selectedfrom AXL, MER and Tyro3.

In some embodiments, the inhibitor agent is capable of antagonizing theminor GAS6/receptor binding interaction, where the receptor is selectedfrom AXL, MER and Tyro3.

In some embodiments, the inhibitor agent is a polypeptide, apolypeptide-carrier fusion, a polypeptide-Fc fusion, apolypeptide-conjugate, a polypeptide-drug conjugate, an antibody, abispecific antibody, an antibody drug conjugate, an antibody fragment,an antibody-related structure, or a combination thereof.

In some embodiments, the inhibitor agent is a natural or syntheticpolypeptide.

In some embodiments, the inhibitor agent is a non-antibody polypeptide.

In some embodiments, the inhibitor agent of the present invention caninclude, for example but is not limited to a darpin, an avimer, anadnectin, an anticalin, an affibody, a maxibody, or other proteinstructural scaffold, or a combination thereof.

In some embodiments, the inhibitor agent is a polypeptide-conjugate orantibody-conjugate.

In some embodiments, the inhibitor agent is a polypeptide-polymerconjugate, where the polymer is selected from PEG, PEG-containingpolymers, degradable polymers, biocompatible polymers, hydrogels, andother polymer structures or a combination thereof.

In some embodiments, the inhibitor agent is a polypeptide, wherein saidpolypeptide comprises a soluble AXL variant polypeptide wherein said AXLvariant polypeptide lacks the AXL transmembrane domain and has at leastone mutation relative to wild-type that increases affinity of the AXLpolypeptide binding to GAS6.

In some embodiments, the inhibitor agent is a polypeptide, wherein saidpolypeptide comprises a soluble MER variant polypeptide wherein said MERvariant polypeptide lacks the MER transmembrane domain and has at leastone mutation relative to wild-type that increases affinity of the MERpolypeptide binding to GAS6.

In some embodiments, the inhibitor agent is a polypeptide, wherein saidpolypeptide comprises a soluble Tyro3 variant polypeptide wherein saidTyro3 variant polypeptide lacks the Tyro3 transmembrane domain and hasat least one mutation relative to wild-type that increases affinity ofthe Tyro3 polypeptide binding to GAS6.

In some embodiments, the inhibitor is an AXL, MER or Tyro3 variantpolypeptide that inhibits binding between a wild-type AXL, MER and/orTyro3 polypeptide and a GAS6 protein in vivo or in vitro.

In some embodiments, the polypeptide lacks a functional fibronectin (FN)domain and/or exhibits increased affinity of the AXL, MER or Tyro3variant polypeptide binding to GAS6 compared to wild-type AXL, MER orTyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain, has more than one Ig1 domain and exhibitsincreased affinity of the AXL, MER or Tyro3 variant polypeptide bindingto GAS6 as compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide has twoIg1 domains. In some embodiments, the polypeptide has three Ig1 domains.

In some embodiments, the AXL, MER or Tyro3 polypeptide lacks thetransmembrane domain, has more than one Ig2 domain and exhibitsincreased affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6as compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide has twoIg2 domains.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL variant polypeptide lacksthe AXL, MER or Tyro3 transmembrane domain, has more than one Ig1domain, more than one Ig2 domain and exhibits increased affinity of theAXL, MER or Tyro3 variant polypeptide binding to GAS6 as compared towild-type AXL, MER or Tyro3.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks afunctional fibronectin (FN) domain, has more than one Ig1 domain, morethan one Ig2 domain, and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidehas two Ig1 domains and two Ig2 domains.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidehas the immunoglobulin domains connected directly.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidehas the immunoglobulin domains connected indirectly.

In some embodiments, the soluble AXL, MER or Tyro3 variant polypeptidelacks the AXL, MER or Tyro3 transmembrane domain, is capable of bindingboth the major and minor binding site of a single GAS6 and wherein saidAXL, MER or Tyro3 variant polypeptide exhibits increased affinity of theAXL, MER or Tyro3 polypeptide binding to GAS6 as compared to wild-typeAXL, MER or Tyro3.

In some embodiments, the polypeptide has one Ig1 domain and lacks afunctional Ig2 domain.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, has oneIg1 domain, lacks a functional Ig2 domain and wherein said AXL, MER orTyro3 variant polypeptide exhibits increased affinity of the AXL, MER orTyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MERor Tyro3.

In some embodiments, the polypeptide is a soluble AXL, MER or Tyro3variant polypeptide, wherein said soluble AXL, MER or Tyro3 variantpolypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks afunctional fibronectin (FN) domain, has one Ig1 domain, lacks afunctional Ig2 domain and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide is afusion protein comprising an Fc domain.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide furthercomprises a linker. In some embodiments, the linker comprises one ormore (GLY)₄SER units.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks theAXL, MER or Tyro3 intracellular domain.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks afunctional fibronectin (FN) domain and wherein said AXL, MER or Tyro3variant polypeptide exhibits increased affinity of the polypeptidebinding to GAS6 as compared to wild-type AXL, MER or Tyro3.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide comprisesat least one amino acid modification relative to the wild-type AXL, MERor Tyro3 sequence.

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification within a region selected from thegroup consisting of 1) between 15-50, 2) between 60-120, and 3) between125-135 of the wild-type AXL sequence (SEQ ID NO:1).

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification at position 19, 23, 26, 27, 32, 33,38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109,112, 113, 116, 118, or 127 of the wild-type AXL sequence (SEQ ID NO: 1)or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide comprises atleast one amino acid modification selected from the group consistingof 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K 5) G32S, 6) N33S, 7) T38I,8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15)Q86R, 16) D87G, 17) D88N, 18) I90M or I90V, 19) V92A, V92G or V92D, 20)I97R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A, 25) F113L, 26)H116R, 27) T118A, 28) G127R or G127E, and 29) G129E and a combinationthereof.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following positions: (a) glycine 32; (b) aspartic acid 87; (c)valine 92; and (d) glycine 127.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following positions: (a) aspartic acid 87 and (b) valine 92.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following positions: (a) glycine 32; (b) aspartic acid 87; (c)valine 92; (d) glycine 127 and (e) alanine 72.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following position: alanine 72.

In some embodiments, in the soluble AXL variant polypeptide the glycine32 residue is replaced with a serine residue, aspartic acid 87 residueis replaced with a glycine residue, valine 92 residue is replaced withan alanine residue, or glycine 127 residue is replaced with an arginineresidue or a combination thereof.

In some embodiments, in the soluble AXL variant polypeptide asparticacid 87 residue is replaced with a glycine residue or valine 92 residueis replaced with an alanine residue or a combination thereof.

In some embodiments, in the soluble AXL variant polypeptide alanine 72residue is replaced with a valine residue.

In some embodiments, in the soluble AXL variant polypeptide glycine 32residue is replaced with a serine residue, aspartic acid 87 residue isreplaced with a glycine residue, valine 92 residue is replaced with analanine residue, glycine 127 residue is replaced with an arginineresidue or an alanine 72 residue is replaced with a valine residue or acombination thereof.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following positions: (a) glutamic acid 26; (b) valine 79; (c) valine92; and (d) glycine 127.

In some embodiments, in the soluble AXL variant polypeptide glutamicacid 26 residue is replaced with a glycine residue, valine 79 residue isreplaced with a methionine residue, valine 92 residue is replaced withan alanine residue, or glycine 127 residue is replaced with an arginineresidue or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide comprises atleast an amino acid region selected from the group consisting of aminoacid region 19-437, 130-437, 19-132, 21-121, 26-132, 26-121 and 1-437 ofthe wild-type AXL polypeptide (SEQ ID NO: 1), and wherein one or moreamino acid modifications occur in said amino acid region.

In some embodiments, the soluble AXL variant polypeptide comprises aminoacid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) atthe following positions: (a) glycine 32; (b) aspartic acid 87; (c)alanine 72; and valine 92.

In some embodiments, in the soluble AXL variant polypeptide glycine 32is replaced with a serine residue, aspartic acid 87 is replaced with aglycine residue, alanine 72 is replaced with a valine residue, andvaline 92 is replaced with an alanine residue, or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain and wherein said AXL variant comprisesamino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) atthe following positions: (a) glycine 32; (b) aspartic acid 87; (c)alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL variant polypeptide of any of thepreceding claims, wherein the soluble AXL polypeptide is a fusionprotein comprising an Fc domain and wherein glycine 32 is replaced witha serine residue, aspartic acid 87 is replaced with a glycine residue,alanine 72 is replaced with a valine residue, and valine 92 is replacedwith an alanine residue, or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain and wherein said AXL variant comprisesamino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) atthe following positions: (a) glycine 32; (b) aspartic acid 87; (c)alanine 72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain and wherein glycine 32 is replaced witha serine residue, aspartic acid 87 is replaced with a glycine residue,alanine 72 is replaced with a valine residue, valine 92 is replaced withan alanine residue, and glycine 127 is replaced with an arginine residueor a combination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, andwherein said AXL variant comprises amino acid changes relative towild-type AXL sequence (SEQ ID NO:1) at the following positions: (a)glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, andwherein glycine 32 is replaced with a serine residue, aspartic acid 87is replaced with a glycine residue, alanine 72 is replaced with a valineresidue, and valine 92 is replaced with an alanine residue, or acombination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, andwherein said AXL variant comprises amino acid changes relative towild-type AXL sequence (SEQ ID NO:1) at the following positions: (a)glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e)glycine 127.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, andwherein glycine 32 is replaced with a serine residue, aspartic acid 87is replaced with a glycine residue, alanine 72 is replaced with a valineresidue, valine 92 is replaced with an alanine residue, and glycine 127is replaced with an arginine residue or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, lacks anIg2 domain, and wherein said AXL variant comprises amino acid changesrelative to wild-type AXL sequence (SEQ ID NO:1) at the followingpositions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72 and (d)valine 92.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, lacks anIg2 domain and wherein glycine 32 is replaced with a serine residue,aspartic acid 87 is replaced with a glycine residue, alanine 72 isreplaced with a valine residue, and valine 92 is replaced with analanine residue or a combination thereof.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, lacks anIg2 domain, and wherein said AXL variant comprises amino acid changesrelative to wild-type AXL sequence (SEQ ID NO:1) at the followingpositions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d)valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant polypeptide is a fusionprotein comprising an Fc domain, lacks a functional FN domain, lacks anIg2 domain and wherein glycine 32 is replaced with a serine residue,aspartic acid 87 is replaced with a glycine residue, alanine 72 isreplaced with a valine residue, valine 92 is replaced with an alanineresidue, and glycine 127 is replaced with an arginine residue or acombination thereof.

In some embodiments, the soluble AXL variant polypeptide of any of thepreceding claims, wherein said soluble AXL variant polypeptide has anaffinity of at least about 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M or1×10⁻¹² M for GAS6.

In some embodiments, the soluble AXL variant polypeptide exhibits anaffinity to GAS6 that is at least about 5-fold stronger, at least about10-fold stronger or at least about 20-fold stronger than the affinity ofthe wild-type AXL polypeptide.

In some embodiments, the soluble AXL variant polypeptide comprises oneor more (GLY)4SER (SEQ ID NO:10) units. In some embodiments, the linkercomprises 1, 2, 3 or 5 (GLY)4SER (SEQ ID NO:10) units.

In some embodiments, the soluble AXL variant polypeptide inhibitsbinding between wild-type AXL, MER and/or Tyro3 polypeptide and a GAS6protein in vivo or in vitro.

In some embodiments, the soluble AXL variant polypeptide is a fusionpolypeptide comprising an Fc domain.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of one or moresoluble AXL, MER or Tyro3 variant polypeptides.

In some embodiments, the pharmaceutical composition further comprises atleast one cytotoxic agent or a pharmaceutically acceptable excipient ora combination thereof.

In some embodiments, the present invention also provides methods oftreating, reducing, or preventing the metastasis or invasion of a tumorin a mammalian patient, the method comprising: administering to saidpatient an effective dose of the inhibitor agent of the presentinvention. In some embodiments, the inhibitor agent is an AXL, MER orTyro3 variant polypeptide of any of the preceding claims.

In some embodiments, the tumor for treatment is a tumor selected fromthe group consisting of an ovarian tumor, a breast tumor, a lung tumor,a liver tumor, a colon tumor, a gallbladder tumor, a pancreatic tumor, aprostate tumor, and glioblastoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Describes the four domains of AXL and some embodiments of thevarious combinations of AXL-Fc constructs that have been made andtested.

FIG. 2. Describes some embodiments of the various combinations ofmonovalent AXL-Fc constructs.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given to such terms, the followingdefinitions are provided.

“Inhibitors,” “activators,” and “modulators” of AXL on metastatic cellsor its ligand GAS6 are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for receptor or ligand binding or signaling, e.g., ligands,receptors, agonists, antagonists, and their homologs and mimetics.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of two or more amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. The terms“antibody” and “antibodies” are used interchangeably herein and refer toa polypeptide capable of interacting with and/or binding to anothermolecule, often referred to as an antigen. Antibodies can include, forexample “antigen-binding polypeptides” or “target-molecule bindingpolypeptides.” Antigens of the present invention can include for exampleany polypeptides described in the present invention.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. All single lettersused in the present invention to represent amino acids are usedaccording to recognized amino acid symbols routinely used in the field,e.g., A means Alanine, C means Cysteine, etc. An amino acid isrepresented by a single letter before and after the relevant position toreflect the change from original amino acid (before the position) tochanged amino acid (after position). For example, A19T means that aminoacid alanine at position 19 is changed to threonine.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” thus encompass individuals havingcancer, including without limitation, adenocarcinoma of the ovary orprostate, breast cancer, glioblastoma, etc., including those who haveundergone or are candidates for resection (surgery) to remove canceroustissue. Subjects may be human, but also include other mammals,particularly those mammals useful as laboratory models for humandisease, e.g. mouse, rat, etc.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. In general, cellsof interest for detection, analysis, classification, or treatment in thepresent application include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Examples of cancerinclude but are not limited to, ovarian cancer, glioblastoma, breastcancer, colon cancer, lung cancer, prostate cancer, hepatocellularcancer, gastric cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer,and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body. Therefore,the present invention contemplates a method of determining the risk offurther growth of one or more cancerous tumors in an organ or body partwhich is not directly connected to the organ of the original canceroustumor and/or any steps in a process leading up to that growth.

Depending on the nature of the cancer, an appropriate patient sample isobtained. As used herein, the phrase “cancerous tissue sample” refers toany cells obtained from a cancerous tumor. In the case of solid tumorswhich have not metastasized, a tissue sample from the surgically removedtumor will typically be obtained and prepared for testing byconventional techniques.

The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents; washed; orenrichment for certain cell populations, such as cancer cells. Thedefinition also includes sample that have been enriched for particulartypes of molecules, e.g., nucleic acids, polypeptides, etc. The term“biological sample” encompasses a clinical sample, and also includestissue obtained by surgical resection, tissue obtained by biopsy, cellsin culture, cell supernatants, cell lysates, tissue samples, organs,bone marrow, blood, plasma, serum, and the like. A “biological sample”includes a sample obtained from a patient's cancer cell, e.g., a samplecomprising polynucleotides and/or polypeptides that is obtained from apatient's cancer cell (e.g., a cell lysate or other cell extractcomprising polynucleotides and/or polypeptides); and a sample comprisingcancer cells from a patient. A biological sample comprising a cancercell from a patient can also include non-cancerous cells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as ovarian cancer. The term “prediction” is used herein torefer to the act of foretelling or estimating, based on observation,experience, or scientific reasoning. In one example, a physician maypredict the likelihood that a patient will survive, following surgicalremoval of a primary tumor and/or chemotherapy for a certain period oftime without cancer recurrence.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure (e.g., radiation,a surgical procedure, etc.), for the purposes of obtaining an effect.The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of effecting a partial or complete cure for a disease and/orsymptoms of the disease. “Treatment,” as used herein, covers anytreatment of any metastatic tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease. In tumor (e.g., cancer)treatment, a therapeutic agent may directly decrease the metastasis oftumor cells.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of an cancer, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withneoplasia, e.g., tumor or cancer. The term “therapeutic effect” refersto the reduction, elimination, or prevention of the disease, symptoms ofthe disease, or side effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

According to the present invention, the first therapeutic can be anysuitable therapeutic agent, e.g., cytotoxic agents. One exemplary classof cytotoxic agents are chemotherapeutic agents, e.g., they can becombined with treatment to inhibit AXL or GAS6 signaling. Exemplarychemotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, duocarmycin, epoetin alpha,etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine,granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha,irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna,methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone,omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine,prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecanhydrochloride, trastuzumab, vinblastine, vincristine and vinorelbinetartrate. For ovarian cancer treatment, a preferred chemotherapeuticagent with which an AXL or GAS6 signaling inhibitor can be combined ispaclitaxel (Taxol™).

Other combination therapies are radiation, surgery, and hormonedeprivation (Kwon et al., Proc. Natl. Acad. Sci U.S.A., 96: 15074-9,1999). Angiogenesis inhibitors can also be combined with the methods ofthe invention.

“Concomitant administration” of a known cancer therapeutic drug with apharmaceutical composition of the present invention means administrationof the drug and AXL inhibitor at such time that both the known drug andthe composition of the present invention will have a therapeutic effect.Such concomitant administration may involve concurrent (i.e. at the sametime), prior, or subsequent administration of the drug with respect tothe administration of a compound of the present invention. A person ofordinary skill in the art would have no difficulty determining theappropriate timing, sequence and dosages of administration forparticular drugs and compositions of the present invention.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the invention.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

DETAILED DESCRIPTION

AXL, MER and Tyro3 are the three receptor protein tyrosine kinases whoseligand is GAS6. As such, the present invention is based in part on thediscovery of inhibitor agents that inhibit and/or antagonize theinteraction of the wild-type AXL, MER and/or Tyro3 receptor with theGAS6 ligand.

According to the present invention, such an inhibitor agent can beselected from (a) an inhibitor of AXL, MER and/or Tyro3 activity, (b) aninhibitor of GAS6 activity and (c) an inhibitor of AXL, MER and/orTyro3-GAS6 interaction, wherein the inhibitor agent is capable ofbinding to GAS6 with increased affinity compared to wild-type AXL, MERand/or Tyro3.

In some embodiments, the inhibitor agent binds to two or more epitopeson a single GAS6 molecule. The two or more epitopes can include at leastone of the major and/or minor AXL, MER and/or Tyro3 binding site onGAS6. In some embodiments, the epitopes are separate or distinctepitopes. In some embodiments the epitopes overlap. In some embodiments,the epitopes do not overlap. In some embodiments, the epitopes areadjacent. In some embodiments, the epitopes are not adjacent. In someembodiments, the epitopes include the major and/or minor AXL, MER and/orTyro3 binding site on GAS6. These GAS6 epitopes of the presentinvention, and to which the inhibitor agents of the present inventionbind, can be located on one or more GAS6 molecules. In some embodiments,the epitopes are located on a single GAS6 molecule.

In some embodiments, the inhibitor agent is capable of binding to themajor and/or minor AXL, MER and/or Tyro3 binding sites on a single GAS6.In some embodiments, the inhibitor agent is capable of binding the majorAXL, MER and/or Tyro3 binding site of GAS6 and one or more additionalGAS6 epitopes. In other embodiments, the inhibitor agent is capable ofbinding to the AXL, MER and/or Tyro3 minor binding site on GAS6 and oneor more additional epitopes. In some other embodiments, the inhibitoragent is capable of binding two or more distinct epitopes on GAS6. Theadditional GAS6 epitopes can include any epitopes on GAS6 which lead toincreased affinity and/or increased avidity of the inhibitor agentbinding to GAS6 as compared to wild-type AXL, MER and/or Tyro3. In someembodiments, the AXL, MER and/or Tyro3 variant polypeptides of thepresent invention bind two epitopes on a single GAS6 molecule. In someembodiments, the two epitopes are the major and minor AXL, MER and/orTyro3 binding sites.

According to the invention, GAS6 receptors include AXL, MER and Tyro3.The inhibitor agents of the present invention can also in someembodiments antagonize the major and/or minor GAS6/receptor interaction.In some embodiments, the inhibitor agent is capable of antagonizing themajor and/or minor GAS6/receptor binding interaction. In otherembodiments, the inhibitor agent is capable of antagonizing the majorGAS6/receptor binding interaction (e.g., interfering with and/orinhibiting the major GAS6/receptor binding interaction). In someembodiments, the inhibitor agent is capable of antagonizing the minorGAS6/receptor binding interaction (e.g., interfering with and/orinhibiting the minor GAS6/receptor binding interaction).

Inhibitor agents can also include for example protein scaffolds (i.e.,smaller proteins that are capable of achieving comparable affinity andspecificity using molecular structures that can be for example one-tenththe size of full antibodies).

The inhibitor agents can also include non-antibody polypeptides. In someembodiments, the inhibitor agent is a non-antibody polypeptide. In someembodiments, the non-antibody polypeptide can include but is not limitedto peptibodies, darpins, avimers, adnectins, anticalins, affibodies,maxibodies, or other protein structural scaffold, or a combinationthereof.

In some embodiments the inhibitor agent provided by the presentinvention is an AXL, MER and/or Tyro3 variant polypeptide, e.g., an AXL,MER and/or Tyro3 variant polypeptide that has a binding activity to GAS6that is substantially equal to or better than the binding activity of awild-type AXL, MER and/or Tyro3 polypeptide. In some embodiments of thepresent invention, the AXL, MER and/or Tyro3 variant polypeptides areutilized as therapeutic agents.

The AXL protein, with reference to the native sequence of SEQ ID NO: 1,comprises an immunoglobulin (Ig)-like domain from residues 27-128, asecond Ig-like domain from residues 139-222, fibronectin type 3 domainsfrom residues 225-332 and 333-427, intracellular domain from residues473-894 including tyrosine kinase domain. The tyrosine residues at 779,821 and 866 become autophosphorylated upon receptor dimerization andserve as docking sites for intracellular signaling molecules. The nativecleavage site to release the soluble form of the polypeptide lies atresidues 437-451.

For the purposes of the invention, a soluble form of AXL (sAXL) is theportion of the polypeptide that is sufficient to bind GAS6 at arecognizable affinity, e.g., high affinity, which normally lies betweenthe signal sequence and the transmembrane domain, i.e. generally fromabout SEQ ID NO: 1 residue 19-437, but which may comprise or consistessentially of a truncated version of from about residue 19, 25, 30, 35,40, 45, 50 to about residue 132, 450, 440, 430, 420, 410, 400, 375, 350,to 321, e.g., residue 19-132. According to the methods of the presentinvention, SEQ ID NO:1 can be used interchangeably with amino acids8-894 of SEQ ID NO: 1, both of which refer to the wild-type AXLsequence. In some embodiments, a soluble form of AXL lacks thetransmembrane domain, and optionally the intracellular domain.

In some embodiments, the inhibitor agent is a soluble AXL variantpolypeptide that lacks the AXL transmembrane domain and has at least onemutation relative to wild-type that increases affinity of the AXLpolypeptide binding to GAS6 as compared to wild-type GAS6.

The MER protein, with reference to the native SEQ ID NO:2, comprises animmunoglobulin (Ig)-like domain from residues 81-186, a second Ig-likedomain from residues 197-273, fibronectin type 3 domains from residues284-379 and 383-482, intracellular domain from residues 527-999including tyrosine kinase domain. The tyrosine residues at 749, 753, 754and 872 become autophosphorylated upon receptor dimerization and serveas docking sites for intracellular signaling molecules.

For the purposes of the invention, a soluble form of MER (sMER) is theportion of the polypeptide that is sufficient to bind GAS6 at arecognizable affinity, e.g., high affinity, which normally lies betweenthe signal sequence and the transmembrane domain, i.e. generally fromabout SEQ ID NO: 2 residue 21-526, but which may comprise or consistessentially of a truncated version In some embodiments, a soluble formof MER lacks the transmembrane domain (i.e., generally from about SEQ IDNO: 2 residue 506-526), and optionally the intracellular domain (i.e.,generally from about SEQ ID NO: 2 residue 527-999).

In some embodiments, the inhibitor agent is a soluble MER variantpolypeptide wherein said MER polypeptide lacks the MER transmembranedomain and has at least one mutation relative to wild-type thatincreases affinity of the MER polypeptide binding to GAS6 as compared towild-type MER.

The Tyro3 protein, with reference to the native SEQ ID NO:3, comprisesan immunoglobulin (Ig)-like domain from residues 41-128, a secondIg-like domain from residues 139-220, fibronectin type 3 domains fromresidues 225-317 and 322-413, intracellular domain from residues 451-890including tyrosine kinase domain. The tyrosine residues at 681, 685, 686and 804 become autophosphorylated upon receptor dimerization and serveas docking sites for intracellular signaling molecules.

For the purposes of the invention, a soluble form of Tyro3 (sTyro3) isthe portion of the Tyro3 polypeptide that is sufficient to bind GAS6 ata recognizable affinity, e.g., high affinity, which normally liesbetween the signal sequence and the transmembrane domain, i.e. generallyfrom about SEQ ID NO: 3 residue 41-450, but which may comprise orconsist essentially of a truncated version In some embodiments, asoluble form of AXL lacks the transmembrane domain (i.e., generally fromabout SEQ ID NO: 3 residue 430-450), and optionally the intracellulardomain (i.e., generally from about SEQ ID NO: 3 residue 451-890).

In some embodiments, the inhibitor agent is a soluble Tyro3 variantpolypeptide wherein said Tyro3 polypeptide lacks the Tyro3 transmembranedomain and has at least one mutation relative to wild-type Tyro3 thatincreases affinity of the Tyro3 polypeptide binding to GAS6 as comparedto wild-type Tyro3.

In some embodiments, the AXL, MET or Tyro3 variant polypeptide lacks theAXL, MET or Tyro3 transmembrane domain and is a soluble variantpolypeptide, e.g., sAXL, sMER or sTyro3 variant polypeptide.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks theAXL, MER or Tyro3 intracellular domain.

In some embodiments, the inhibitor agent of the present inventioninhibits binding between a wild-type AXL, MER and/or Tyro3 polypeptideand a GAS6 protein in vivo or in vitro. In some embodiments, the AXL,MER or Tyro3 variant polypeptide inhibits binding between a wild-typeAXL, MER and/or Tyro3 polypeptide and a GAS6 protein in vivo or invitro.

The inhibitor agents of the present invention can also exhibit anenhanced or better pharmacokinetic profile. In some embodiments, theenhanced or better pharmacokinetic profile includes for example but isnot limited to a better absorption profile, better distribution profile,better metabolism profile, better excretion profile, better liberationprofile, increased half-life, decrease half-life, faster rate of action,longer duration of effect as compared to AXL, MER and/or Tyro3 wild-typepolypeptides which do not lack a transmembrane domain. One of skill inthe art would understand preferred pharmacokinetic profile parametersfor particular needs including for example treatment regimens, and howto appropriately implement such parameters in treatment regimens.

The wild-type AXL, MER and Tyro3 all contain two fibronectin domains. Insome embodiments, the AXL, MER and Tyro3 polypeptides of the inventionlack a functional fibronectin (FN) domain. Lacks or lacking a functionalfibronectin domain can include but is not limited to deletion of one orboth fibronectin domains and/or introducing mutations that inhibit,reduce or remove the functionality of one or both fibronectin domains,where such mutations can include for example but are not limited tosubstitution, deletion and insertion mutations. In some embodiments, thepolypeptides of the invention have fibronectin 1 (FN1) deleted,fibronectin 2 (FN2) deleted, or FN1 and FN2 both deleted. In someembodiments, the polypeptides of the invention have portions of FN1mutated and/or deleted, FN2 mutated and/or deleted, or FN1and FN2mutated and/or deleted.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks afunctional AXL, MER or Tyro3 fibronectin (FN) domain. In someembodiments, the AXL, MER or Tyro3 variant polypeptide exhibitsincreased affinity of the polypeptide binding to GAS6 as compared towild-type AXL, MER and/or Tyro3. In some embodiments, the AXL, MER orTyro3 variant polypeptide lacks a functional fibronectin (FN) domainalso exhibits increased affinity of the polypeptide binding to GAS6 ascompared to wild-type AXL, MER and/or Tyro3.

In some embodiments, the lack of a functional fibronectin domain resultsin increased affinity of the AXL, MER or Tyro3 polypeptide binding toGAS6. In some embodiments, the lack of a functional fibronectin domainresults in an enhanced or better pharmacokinetic profile, including forexample but not limited to a better absorption profile, betterdistribution profile, better metabolism profile, better excretionprofile, better liberation profile, increased half-life, decreasedhalf-life, faster rate of action, longer duration of effect as comparedto other wild-type polypeptides or other polypeptides which do not lacka functional fibronectin domain. One of skill in the art wouldunderstand preferred pharmacokinetic profile parameters for particularneeds including for example treatment regimens, and how to appropriatelyimplement such parameters in treatment regimens.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain and has more than one Ig1 domain and exhibitsincreased affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6as compared to wild-type AXL, MER and/or Tyro3. In some embodiments, theAXL, MER or Tyro3 polypeptide has two Ig1 domains. In some embodiments,the AXL, MER or Tyro3 polypeptide has three Ig1 domains. In someembodiments, the AXL, MER or Tyro3 polypeptide has more than one Ig1domain and/or more than one Ig2 domain. In some embodiments, the AXL,MER or Tyro3 polypeptide has two Ig2 domains. In some embodiments, theAXL, MER or Tyro3 polypeptide has two Ig1 domains and 2 Ig2 domains. Insome embodiments, the AXL, MER or Tyro3 polypeptide includes for examplebut is not limited to one of the following Ig domain configurations, aswell as any combinations or variations thereof:

-   -   Ig1    -   Ig1-Ig2    -   Ig1-Ig1    -   Ig1-Ig1-Ig1    -   Ig1-Ig2-Ig1    -   Ig1-Ig2-Ig 1-Ig2

In some embodiments, the AXL, MER or Tyro3 polypeptide also lacks theAXL, MER or Tyro3 transmembrane domain and/or exhibits increasedaffinity of the AXL, MER or Tyro3 polypeptide binding to GAS6. In someembodiments, the AXL, MER or Tyro3 variant polypeptide lacks thetransmembrane domain, has more than one Ig1 domain, has more than oneIg2 domain and exhibits increased affinity of the AXL, MER or Tyro3polypeptide binding to GAS6 as compared to wild-type AXL, MER and/orTyro3.

In some embodiments, the AXL, MER or Tyro3 has the immunoglobulindomains connected directly to one another. In some embodiments, the AXL,MER or Tyro3 has the immunoglobulin domains connected indirectly, e.g.,through a linker molecule including for example any amino acid linkerknown in the art.

In some embodiments, the one or more AXL, MER or Tyro3 Ig1 and/or 1 ormore AXL, MER or Tyro3 Ig2 domains result in an enhanced or betterpharmacokinetic profile, including for example but not limited to abetter absorption profile, better distribution profile, bettermetabolism profile, better excretion profile, better liberation profile,increased half-life, decreased half-life, faster rate of action, longerduration of effect as compared to other wild-type polypeptides or otherpolypeptides which do not lack a functional fibronectin domain. One ofskill in the art would understand preferred pharmacokinetic profileparameters for particular needs including for example treatmentregimens, and how to appropriately implement such parameters intreatment regimens.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide lacks theAXL, MER or Tyro3 transmembrane domain and is capable of binding two ormore epitopes on a single GAS6. In some embodiments, the AXL, MER orTyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembranedomain and is capable of binding both the major and minor AXL, MERand/or Tyro3 binding sites on a single GAS6. In some embodiments, thebinding of both the major and minor AXL, MER and/or Tyro3 binding issimultaneous. In some embodiments, the binding of both the major andminor AXL, MER and/or Tyro3 binding sites is simultaneous on a singleGAS6.

The present invention also provides AXL, MER or Tyro3 variantpolypeptides that do not bind two epitopes on a single GAS6 molecule.The present invention also provides AXL, MER or Tyro3 variantpolypeptides that do not bind two epitopes on a single GAS6 moleculesimultaneously. In some embodiments, the AXL, MER and/or Tyro3 variantpolypeptide is not capable of binding two epitopes on a single GAS6,this includes for example monomeric AXL, MER and/or Tyro3 variantpolypeptides. In some embodiments, the monomeric AXL, MER or Tyro3variant polypeptide comprises one Ig1 domain. In some embodiments, themonomeric AXL, MER and/or Tyro3 variant polypeptide is an Fc fusionpolypeptide that does not bind to more than one site on a singe Gas6molecule simultaneously. In some embodiments, the monomeric AXL, MERand/or Tyro3 variant polypeptide that is not capable of binding twoepitopes on a single GAS6 comprises two AXL, MER and/or Tyro3 variantpolypeptides each of which are not capable of binding two epitopes on asingle GAS6 simultaneously. In some embodiments, the monomeric AXL, MERand/or Tyro3 variant polypeptide that is not capable of simultaneouslybinding two epitopes on a single GAS6 has one Ig1 domain. In someembodiments, the monomeric AXL, MER and/or Tyro3 variant polypeptidethat is not capable of simultaneously binding two epitopes on a singleGAS6 has an altered half-life when compared to AXL, MER and/or Tyro3variant polypeptides that are capable of binding two epitopes on asingle GAS6. In some embodiments, the polypeptide has one Ig1 domain andlacks a functional Ig2 domain. In some embodiments, the Ig1 domaincomprises amino acids 1-131 of AXL (SEQ ID NO:1; or in some embodiments8-138 of SEQ ID NO:1). In some embodiments, the polypeptide is a solubleAXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER orTyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembranedomain, has one Ig1 domain, lacks a functional Ig2 domain and whereinsaid AXL, MER or Tyro3 variant polypeptide exhibits increased affinityof the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared towild-type AXL, MER or Tyro3. In some embodiments, the polypeptide of anyof the preceding claims, wherein the polypeptide is a soluble AXL, MERor Tyro3 variant polypeptide, wherein said soluble AXL, MER or Tyro3variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain,lacks a functional fibronectin (FN) domain, has one Ig1 domain, lacks afunctional Ig2 domain and wherein said AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3 variantpolypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3.

The wild-type AXL, MER and Tyro3 all contain an Ig2 domain. In someembodiments, the AXL, MER and Tyro3 polypeptides of the invention lack afunctional Ig2 domain. Lacks or lacking a functional Ig2 domain caninclude but is not limited to deletion of the Ig2 domain and/orintroduction of mutations that inhibit, reduce or remove thefunctionality of the Ig2 domain, where such mutations can include forexample but are not limited to substitution, deletion and insertionmutations. In some embodiments, the polypeptides of the invention lack afunctional Ig2 domain. In some embodiments, the polypeptides of theinvention lack a functional Ig2 domain and have a wild-type AXL, MERand/or Tyro3 Ig1 domain. In some embodiments, the polypeptides of theinvention lack a functional Ig2 domain and have one or more mutations inthe Ig1 domain relative to the wild-type AXL, MER and/or Tyro3 Ig1domain.

In some embodiments, the AXL, MER and/or Tyro3 variant polypeptideincludes a linker. A wide variety of linkers are known in the art andany known linker can be employed with the methods of the presentinvention. In some embodiments, the AXL, MER or Tyro3 variantpolypeptide includes one or more linkers or linker units. In someembodiments, the linker is an amino acid linker, including an amino acidsequence of 2, 3, 4 or 5 amino acids which are different that thewild-type AXL, MER and/or Tyro3 sequences. In some embodiments, thelinker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more units. In someembodiments, the linker is (GLY)4SER (SEQ ID NO:10). In someembodiments, the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more(GLY)4SER (SEQ ID NO:10) units. In some embodiments, the linker has 1,2, 3 or 5 (GLY)4SER (SEQ ID NO:10) units. In some embodiments, thelinkers are between the AXL, MER or Tyro3 variant polypeptide and the Fcportion of a fusion polypeptide. In some embodiments, the linkers arebetween the AXL, MER or Tyro3 variant polypeptide and the Fc portion ofa fusion polypeptide and the AXL, MER or Tyro3 variant polypeptide lacksa functional fibronectin domain.

In some embodiments, AXL, MER and/or Tyro3 variant polypeptides of thepresent invention also include one or more amino acid modificationswithin the soluble form of wild-type AXL, MER and/or Tyro3, e.g., one ormore amino acid modifications that increase its affinity for GAS6.According to the present invention, amino acid modifications include anynaturally occurring or man-made amino acid modifications known or laterdiscovered in the field. In some embodiments, amino acid modificationsinclude any naturally occurring mutation, e.g., substitution, deletion,addition, insertion, etc. In some other embodiments, amino acidmodifications include replacing existing amino acid with another aminoacid, e.g., a conservative equivalent thereof. In yet some otherembodiments, amino acid modifications include replacing one or moreexisting amino acids with non-natural amino acids or inserting one ormore non-natural amino acids. In still some other embodiments, aminoacid modifications include at least 1, 2, 3, 4, 5, or 6 or 10 amino acidmutations or changes.

In some exemplary embodiments, one or more amino acid modifications canbe used to alter properties of the soluble form of AXL, MER and/or Tyro3e.g., affecting the stability, binding activity and/or specificity, etc.Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for scanning mutations may be found in Gustin et al.,Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli etal., Mol Gen Genet 199:537-9 (1985); and Prentki et al., Gene 29:303-13(1984). Methods for site specific mutagenesis can be found in Sambrooket al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al.,Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990);Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem177:120-4 (1989).

In some embodiments, AXL variant polypeptides of the present inventioninclude one or more amino acid modifications within one or more regionsof residue 18 to 130, residue 10 to 135, residue 15 to 45, residue 60 to65, residue 70 to 80, residue 85 to 90, residue 91 to 99, residue 104 to110, residue 111 to 120, residue 125 to 130, residue 19 to 437, residue130 to 437, residue 19 to 132, residue 21 to 132, residue 21 to 121,residue 26 to 132, or residue 26 to 121 of wild-type AXL (SEQ ID NO: 1).In some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications within one ormore regions of residue 20 to 130, residue 37 to 124 or residue 141 to212 of wild-type AXL (SEQ ID NO: 1). In yet some other embodiments, AXLpolypeptide variants of the present invention include one or more aminoacid modifications at one or more positions of position 19, 23, 26, 27,32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105,109, 112, 113, 116, 118, 127, or 129 of wild-type AXL (SEQ ID NO 1).

In yet some other embodiments, AXL polypeptide variants of the presentinvention include one or more amino acid modifications including withoutany limitation 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6)N33S, 7) T38I, 8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E,14) V79M, 15) Q86R, 16) D87G, 17) D88N, 18) I90M or I90V, 19) V92A, V92Gor V92D, 20) I97R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A,25) F113L, 26) H116R, 27) T118A, 28) G127R or G127E, and 29) E129K and acombination thereof.

In yet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 32,87, 92, or 127 of wild-type AXL (SEQ ID NO: 1) or a combination thereof,e.g., G32S; D87G; V92A and/or G127R. In yet some other embodiments, AXLpolypeptide variants of the present invention include one or more aminoacid modifications at position 26, 79, 92, 127 of wild-type AXL (SEQ IDNO: 1) or a combination thereof, e.g., E26G, V79M; V92A and/or G127E. Inyet some other embodiments, AXL variant polypeptides of the presentinvention include one or more amino acid modifications at position 32,87, 92, 127 and/or 72 of wild-type AXL (SEQ ID NO: 1) or a combinationthereof, e.g., G32S; D87G; V92A; G127R and/or A72V. In yet some otherembodiments, AXL variant polypeptides of the present invention includeone or more amino acid modifications at position 87, 92 and/or 127 ofwild-type AXL (SEQ ID NO: 1) or a combination thereof, e.g., D87G; V92A;and/or G127R. In yet some other embodiments, AXL variant polypeptides ofthe present invention include one or more amino acid modifications atposition 32, 92, and/or 127 of wild-type AXL (SEQ ID NO: 1) or acombination thereof, e.g., G32S; V92A; and/or G127R. In yet some otherembodiments, AXL variant polypeptides of the present invention includeone or more amino acid modifications at position 32, 87 and/or 127 ofwild-type AXL (SEQ ID NO: 1) or a combination thereof, e.g., G32S; D87G;and/or G127R. In yet some other embodiments, AXL polypeptide variants ofthe present invention include one or more amino acid modifications atposition 32, 87 and/or 92 of wild-type AXL (SEQ ID NO: 1) or acombination thereof, e.g., G32S; D87G; and/or V92A. In yet some otherembodiments, AXL variant polypeptides of the present invention includeone or more amino acid modifications at position 26, 79, 92, 127 ofwild-type AXL (SEQ ID NO: 1) or a combination thereof, e.g., E26G, V79M;V92A and/or G127E. In yet some other embodiments, AXL variantpolypeptides of the present invention include one or more amino acidmodifications at position 87 and 92 of wild-type AXL (SEQ ID NO: 1) or acombination thereof, e.g., D87G and V92A. In yet some other embodiments,AXL variant polypeptides of the present invention include at least oneamino acid modification at position 72 of wild-type AXL (SEQ ID NO: 1),e.g., A72V.

According to the present invention, the inhibitor agent can include butis not limited to a polypeptide, a polypeptide-carrier fusion, apolypeptide-Fc fusion, polypeptide-conjugate, a polypeptide-drugconjugate, an antibody, a bispecific antibody, an antibody-drugconjugate, an antibody fragment, an antibody-related structure, or acombination thereof.

The inhibitor agents of the present invention can include peptides orpolypeptides. The peptides and polypeptides of the present invention caninclude natural and/or synthetic polypeptides. Synthetic polypeptidesand methods of making synthetic polypeptides are well known in the artand any known methods for making synthetic polypeptides can be employedwith the methods of the present invention. In some embodiments, theinhibitor agent is a natural or synthetic polypeptide. In someembodiments, the inhibitor agent is a natural or syntheticpolypeptide-fusion. In some embodiments, the inhibitor agent is anatural or synthetic polypeptide-Fc fusion. In some embodiments thenatural or synthetic polypeptide-fusion is a fusion with another proteinstructural class or scaffold or a natural or syntheticpolypeptide-fusion with a polymer or hydrogel or related structure.

According to the present invention, the AXL, MER or Tyro3 variantpolypeptides of the present invention can be further modified, e.g.,joined to a wide variety of other oligopeptides or proteins for avariety of purposes. For instance, various post-translation orpost-expression modifications can be carried out with respect to AXL,MER or Tyro3 variant polypeptides of the present invention. For example,by employing the appropriate coding sequences, one may providefarnesylation or prenylation. In some embodiments, the AXL, MER or Tyro3variant polypeptides of the present invention can be PEGylated, wherethe polyethyleneoxy group provides for enhanced lifetime in the bloodstream. The AXL, MER or Tyro3 variant polypeptides of the presentinvention can also be combined with other proteins, such as the Fc of anIgG isotype, which can be complement binding, with a toxin, such asricin, abrin, diphtheria toxin, or the like, or with specific bindingagents that allow targeting to specific moieties on a target cell. Theinhibitor agents of the present invention can include polypeptideconjugates and antibody-conjugates. In some embodiments, the inhibitoragent is a polypeptide-conjugate or antibody-conjugate. In someembodiments, the polypeptide conjugate is a drug conjugate. In someembodiments, the peptide or polypeptide conjugate is an antibody-drugconjugates. In some embodiments, the polypeptide conjugate is a polymerconjugate. Polymers of the present invention include but are not limitedto PEG, PEG-containing polymers, degradable polymers, biocompatiblepolymers, hydrogels, as well as other polymer structures that could beconjugated to a polypeptide, and can include combinations thereof.

In some embodiments, the AXL, MER or Tyro3 variant polypeptide of thepresent invention is a fusion protein, e.g., fused in frame with asecond polypeptide. In some embodiments, the second polypeptide iscapable of increasing the size of the fusion protein, e.g., so that thefusion protein will not be cleared from the circulation rapidly. In someother embodiments, the second polypeptide is part or whole of Fc region.In some other embodiments, the second polypeptide is any suitablepolypeptide that is substantially similar to Fc, e.g., providingincreased size and/or additional binding or interaction with Igmolecules. In yet some other embodiments, the second polypeptide is partor whole of an albumin protein, e.g., a human serum albumin protein. Insome embodiments, the second polypeptide is a protein or peptide thatbinds to albumin.

In some other embodiments, the second polypeptide is useful for handlingthe AXL, MER or Tyro3 variant polypeptides, e.g., purification of AXL,MER or Tyro3 variant polypeptides or for increasing stability in vitroor in vivo. For example, AXL, MER or Tyro3 variant polypeptides of thepresent invention can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric or fusion polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. EP A 394,827; Traunecker et al., Nature, 331:84-86, 1988. Fusion proteins having disulfide-linked dimeric structures(due to the IgG) can also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

In yet some other embodiments, the second polypeptide is a markersequence, such as a peptide which facilitates purification of the fusedpolypeptide. For example, the marker amino acid sequence can be ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. Wilson et al., Cell 37: 767, 1984.

In still some other embodiments, the second polypeptide is an entityuseful for improving the characteristics of AXL, MER or Tyro3polypeptide variants of the present invention. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the AXL,MER or Tyro3 polypeptide variants of the present invention to facilitatepurification and subsequently removed prior to final preparation of thepolypeptide. The addition of peptide moieties to facilitate handling ofpolypeptides are familiar and routine techniques in the art.

In still yet some embodiments, AXL, MER or Tyro3 variant polypeptides ofthe present invention have a binding activity to GAS6 that is at leastequal or better than the wild-type AXL, MER or Tyro3. In some otherembodiments, AXL, MER or Tyro3 variant polypeptides of the presentinvention has a binding activity or affinity to GAS6 that is at least1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold greater than that ofthe wild-type AXL, MER or Tyro3. In some other embodiments, AXL, MER orTyro3 polypeptide variant of the present invention has a bindingactivity or affinity to GAS6 of at least about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸ or1×10⁻⁹ M 1×10⁻¹⁰M, 1×10⁻¹¹M or 1×10⁻¹²M. In yet some other embodiments,sAXL polypeptides of the present invention is capable of inhibiting,inhibit or compete with wild-type AXL binding to GAS6 either in vivo, invitro or both. In yet some other embodiments, sAXL polypeptides of thepresent invention inhibit or compete with the binding of AXL S6-1, AXLS6-2, and/or AXL S6-5 (as described in WO2011/091305). In yet some otherembodiments, sAXL polypeptides of the present invention inhibit orcompete with the binding of any sAXL variant as described inWO2011/091305.

The inhibitor agents of the present invention bind to GAS6 withincreased affinity. In some embodiments, the AXL, MER or Tyro3 variantpolypeptide exhibits increased affinity of the AXL, MER or Tyro3polypeptide binding to GAS6 as compared to wild-type AXL, MER or Tyro3.In some embodiments, AXL, MER or Tyro3 variant polypeptide exhibits anaffinity to GAS6 that is at least about 5-fold stronger, at least about10-fold stronger or at least about 20-fold stronger, 50-fold stronger,100-fold stronger or at least 200-fold stronger, etc. than the affinityof the wild-type AXL, MER or Tyro3 polypeptide. In some embodiments, thesoluble AXL has a about a 115-fold stronger affinity to GAS6 than theaffinity of the wild-type AXL polypeptide.

The ability of a molecule to bind to GAS6 can be determined, forexample, by the ability of the putative ligand to bind to GAS6 coated onan assay plate. In one embodiment, the binding activity of AXL, MER orTyro3 variant polypeptides of the present invention to a GAS6 can beassayed by either immobilizing the ligand, e.g., GAS6 or the AXL, MER orTyro3 variant polypeptides. For example, the assay can includeimmobilizing GAS6 fused to a His tag onto Ni-activated NTA resin beads.Agents can be added in an appropriate buffer and the beads incubated fora period of time at a given temperature. After washes to remove unboundmaterial, the bound protein can be released with, for example, SDS,buffers with a high pH, and the like and analyzed.

In still yet other embodiments, AXL, MER or Tyro3 variant polypeptidesof the present invention has a better thermal stability than the thermalstability of a wild-type AXL. In some embodiments, the meltingtemperature of AXL, MER or Tyro3 variant polypeptides of the presentinvention is at least 5° C., 10° C., 15° C., or 20° C. higher than themelting temperature of a wild-type AXL.

According to the present invention, AXL, MER or Tyro3 variantpolypeptides of the present invention can also include one or moremodifications that do not alter primary sequences of the AXL, MER orTyro3 variant polypeptides of the present invention. For example, suchmodifications can include chemical derivatization of polypeptides, e.g.,acetylation, amidation, carboxylation, etc. Such modifications can alsoinclude modifications of glycosylation, e.g. those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g. by exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. In some embodiments, AXL, MERor Tyro3 polypeptide variants of the present invention include AXL, MERor Tyro3 variant polypeptides having phosphorylated amino acid residues,e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

In some other embodiments, AXL, MER or Tyro3 variant polypeptides of thepresent invention include AXL, MER or Tyro3 variant polypeptides furthermodified to improve their resistance to proteolytic degradation or tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, AXL, MER or Tyro3 polypeptide variantsof the present invention further include analogs of AXL, MER or Tyro3variant polypeptides containing residues other than naturally occurringL-amino acids, e.g. D-amino acids or non-naturally occurring syntheticamino acids. D-amino acids may be substituted for some or all of theamino acid residues.

In yet some other embodiments, AXL, MER or Tyro3 variant polypeptides ofthe present invention include at least two same or different AXL, MER orTyro3 variant polypeptides linked covalently or non-covalently. Forexample, in some embodiments, AXL, MER or Tyro3 polypeptide variants ofthe present invention include two, three, four, five, or six same ordifferent AXL, MER or Tyro3 variant polypeptides linked covalently,e.g., so that they will have the appropriate size, but avoiding unwantedaggregation.

According to the present invention, AXL, MER or Tyro3 variantpolypeptides of the present invention can be produced by any suitablemeans known or later discovered in the field, e.g., produced fromeukaryotic or prokaryotic cells, synthesized in vitro, etc. Where theprotein is produced by prokaryotic cells, it may be further processed byunfolding, e.g. heat denaturation, DTT reduction, etc. and may befurther refolded, using methods known in the art.

The AXL, MER or Tyro3 variant polypeptides may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman,etc. By using synthesizers, naturally occurring amino acids may besubstituted with unnatural amino acids. The particular sequence and themanner of preparation will be determined by convenience, economics,purity required, and the like.

The AXL, MER or Tyro3 variant polypeptides may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. For the most part, the compositions which are used willcomprise at least 20% by weight of the desired product, more usually atleast about 75% by weight, preferably at least about 95% by weight, andfor therapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized. One of skill in the art can readily utilize well-knowncodon usage tables and synthetic methods to provide a suitable codingsequence for any of the polypeptides of the invention. Direct chemicalsynthesis methods include, for example, the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. While chemical synthesis of DNA isoften limited to sequences of about 100 bases, longer sequences can beobtained by the ligation of shorter sequences. Alternatively,subsequences may be cloned and the appropriate subsequences cleavedusing appropriate restriction enzymes.

The nucleic acids may be isolated and obtained in substantial purity.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which it is not normally associated on a naturally occurringchromosome. The nucleic acids of the invention can be provided as alinear molecule or within a circular molecule, and can be providedwithin autonomously replicating molecules (vectors) or within moleculeswithout replication sequences. Expression of the nucleic acids can beregulated by their own or by other regulatory sequences known in theart. The nucleic acids of the invention can be introduced into suitablehost cells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

In some embodiments, the present invention provides expression vectorsfor in vitro or in vivo expression of one or more AXL, MER and/or Tyro3polypeptide variants of the present invention, either constitutively orunder one or more regulatory elements. In some embodiments, the presentinvention provides a cell population comprising one or more expressionvectors for expressing AXL, MER and/or Tyro3 polypeptide variants of thepresent invention, either constitutively or under one or more regulatoryelements.

According to another aspect of the invention, it provides isolatedantibodies or fragments thereof which specifically bind to a GAS6protein. GAS6 (growth arrest-specific 6) belongs structurally to thefamily of plasma vitamin K-dependent proteins. GAS6 has a highstructural homology with the natural anticoagulant protein S, sharingthe same modular composition and having 40% sequence identity. GAS6 hasgrowth factor-like properties through its interaction with receptortyrosine kinases of the TAM family; Tyro3, AXL and MER. Human GAS6 is a678 amino acid protein that consists of a gamma-carboxyglutamate(Gla)-rich domain that mediates binding to phospholipid membranes, fourepidermal growth factor-like domains, and two laminin G-like (LG)domains. The sequence of the transcript variants of human GAS6 may beaccessed at Genbank at NM_001143946.1; NM_001143945.1; and NM_000820.2,respectively.

GAS6 employs a unique mechanism of action, interacting through itsvitamin K-dependent GLA (gamma-carboxyglutamic acid) module withphosphatidylserine-containing membranes and through its carboxy-terminalLamG domains with the TAM membrane receptors.

According to the present invention, isolated antibodies of the presentinvention include any isolated antibodies with a recognizable bindingspecificity against GAS6. In some embodiments, isolated antibodies arepartially or fully humanized antibodies. In some other embodiments,isolated antibodies are monoclonal or polyclonal antibodies. In yet someother embodiments, isolated antibodies are chimeric antibodies, e.g.,with consistent regions, variable regions and/or CDR3 or a combinationthereof from different sources. In yet some other embodiments, isolatedantibodies are a combination of various features described herein.

According to the present invention, fragments of the isolated antibodiesof the present invention include a polypeptide containing a region ofthe antibody (either in the context of an antibody scaffold or anon-antibody scaffold) that is sufficient or necessary for arecognizable specific binding of the polypeptide towards GAS6. In someembodiments, fragments of the isolated antibodies of the presentinvention include variable light chains, variable heavy chains, one ormore CDRs of heavy chains or light chains or combinations thereof, e.g.,Fab, Fv, etc. In some embodiments, fragments of the isolated antibodiesof the present invention include a polypeptide containing a single chainantibody, e.g., ScFv. In yet some embodiments, fragments of the isolatedantibodies of the present invention include variable regions only orvariable regions in combination with part of Fc region, e.g., CH1region. In still some embodiments, fragments of the isolated antibodiesof the present invention include minibodies, e.g., VL-VH-CH3 ordiabodies.

In some embodiments, isolated antibodies of the present invention bindto an epitope comprised in or presented by one or more amino acidregions that interact with AXL, MER and/or Tyro3. In some otherembodiments, isolated antibodies of the present invention bind to anepitope comprised in or presented by one or more amino acid regions ofGAS6, e.g., L295-T317, E356-P372, R389-N396, D398-A406, E413-H429, andW450-M468 of GAS6.

In yet some other embodiments, isolated antibodies of the presentinvention bind to an epitope comprised in or presented by one or moreamino acid regions, e.g., LRMFSGTPVIRLRFKRLQPT (SEQ ID NO: 4),EIVGRVTSSGP (SEQ ID NO: 5), RNLVIKVN (SEQ ID NO: 6), DAVMKIAVA (SEQ IDNO: 7), ERGLYHLNLTVGIPFH (SEQ ID NO: 8), and WLNGEDTTIQETVVNRM (SEQ IDNO: 9).

In yet some other embodiments, isolated antibodies of the presentinvention bind to an epitope comprised in or presented by at least one,two, three, four, five, or six amino acids in a region of L295-T317,E356-P372, R389-N396, D398-A406, E413-H429, and W450-M468 of GAS6. Inyet some other embodiments, isolated antibodies of the present inventionbind to an epitope comprised in or presented by at least one, two,three, four, five or six amino acids in a region of LRMFSGTPVIRLRFKRLQPT(SEQ ID NO:4), EIVGRVTSSGP (SEQ ID NO:5), RNLVIKVN (SEQ ID NO: 6),DAVMKIAVA (SEQ ID NO: 7), ERGLYHLNLTVGIPFH (SEQ ID NO: 8), andWLNGEDTTIQETVVNRM (SEQ ID NO: 9).

In still some other embodiments, isolated antibodies of the presentinvention is capable of inhibiting, inhibits or competes with thebinding between wild-type AXL, MER and/or Tyro3 or AXL, MER and/or Tyro3polypeptide variants of the present invention and GAS6.

According to the present invention, the AXL, MER or Tyro3 variantpolypeptides and isolated antibodies of the present invention can beprovided in pharmaceutical compositions suitable for therapeutic use,e.g., for human treatment. In some embodiments, pharmaceuticalcompositions of the present invention include one or more therapeuticentities of the present invention, e.g., AXL polypeptide variants and/orisolated antibodies against GAS6 or pharmaceutically acceptable salts,esters or solvates thereof or any prodrug thereof. In some otherembodiments, pharmaceutical compositions of the present inventioninclude one or more therapeutic entities of the present invention incombination with another cytotoxic agent, e.g., another anti-tumoragent. In yet some other embodiments, pharmaceutical compositions of thepresent invention include one or more therapeutic entities of thepresent invention in combination with another pharmaceuticallyacceptable excipient.

In still some other embodiments, therapeutic entities of the presentinvention are often administered as pharmaceutical compositionscomprising an active therapeutic agent, i.e., and a variety of otherpharmaceutically acceptable components. (See Remington's PharmaceuticalScience, 15.sup.th ed., Mack Publishing Company, Easton, Pa., 1980). Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In still some other embodiments, pharmaceutical compositions of thepresent invention can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

According to yet another aspect of the invention, it provides methodsfor treating, reducing or preventing tumor metastasis or tumor invasionby inhibiting the AXL, MER or Tyro3 signaling pathway and/or GAS6signaling pathway. In some embodiments, methods of the present inventioninclude inhibiting the activity of AXL, MER, Tyro3 and/or GAS6, or theinteraction between AXL, MER and/or Tyro3 and GAS6. For example, theactivity of AXL, MER, Tyro3 and/or GAS6 can be inhibited at the geneexpression level, mRNA processing level, translation level,post-translation level, protein activation level, etc. In some otherexamples, the activity of AXL, MER, Tyro3 or GAS6 can be inhibited bysmall molecules, biological molecules, e.g., polypeptides,polynucleotides, antibodies, antibody drug conjugates, etc. In someother examples, the activity of AXL, MER, Tyro3 or GAS6 can be inhibitedby one or more AXL, MER or Tyro3 variant polypeptides or isolatedantibodies of the present invention.

In yet other embodiments, methods of the present invention includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of a therapeutic entity (e.g.,inhibitor agent) of the present invention, e.g., an inhibitor of AXL,MER and/or Tyro3 activity or GAS6 activity or an inhibitor ofinteraction between AXL, MER and/or Tyro3 and GAS6. In some embodiments,effective doses of the therapeutic entity of the present invention, e.g.for the treatment of metastatic cancer, described herein vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

In some embodiments, the dosage may range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. Therapeutic entities of the present invention are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present inventioncan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor metastasis or tumor invasion ofovarian cancer, breast cancer, lung cancer, liver cancer, colon cancer,gallbladder cancer, pancreatic cancer, prostate cancer, and/orglioblastoma.

In still yet some other embodiments, for prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of a disease or condition in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease.

In still yet some other embodiments, for therapeutic applications,therapeutic entities of the present invention are administered to apatient suspected of, or already suffering from such a disease in anamount sufficient to cure, or at least partially arrest, the symptoms ofthe disease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient response has been achieved. Typically, the response ismonitored and repeated dosages are given if there is a recurrence of thecancer.

According to the present invention, compositions for the treatment ofmetastatic cancer can be administered by parenteral, topical,intravenous, intratumoral, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal or intramuscular means. Themost typical route of administration is intravenous or intratumoralalthough other routes can be equally effective.

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. I n general, glycols such as propylene glycol or polyethyleneglycol are preferred liquid carriers, particularly for injectablesolutions. Antibodies and/or polypeptides can be administered in theform of a depot injection or implant preparation which can be formulatedin such a manner as to permit a sustained release of the activeingredient. An exemplary composition comprises polypeptide at 1 mg/mL,formulated in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose,51 mM L-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl orNaOH.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998.Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24,1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Preferably, a therapeutically effective dose of the antibodycompositions described herein will provide therapeutic benefit withoutcausing substantial toxicity.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch. 1).

Also within the scope of the invention are kits comprising thecompositions (e.g., AXL, MER or Tyro3 variant polypeptides andformulations thereof) of the invention and instructions for use. The kitcan further contain a least one additional reagent. Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

According to yet another aspect of the invention, it provides methodsfor determining the ability of a tumor to undergo tumor invasion and/ormetastasis by detecting and/or determining the level of AXL, MER and/orTyro3 activity or GAS6 activity in a biological sample from a subject ofinterest. In some embodiment, the level of AXL, MER and/or Tyro3activity or GAS6 activity is measured by the level of mRNA expression,the level of protein expression, the level of protein activation or anysuitable indicator corresponding to the activity of AXL, MER and/orTyro3 or GAS6 either directly or indirectly. In some embodiments, thelevel of AXL, MER and/or Tyro3 activity or GAS6 activity in a biologicalsample is further compared to a predetermined level, e.g., standardlevel obtained by establishing normal levels or ranges of AXL, MERand/or Tyro3 activity or GAS6 activity based on a population of samplesfrom tumors that do not develop tumor invasion or tumor metastasis orfrom normal tissues. For example, an increase of AXL, MER and/or Tyro3activity or GAS6 activity over the predetermined level or standard levelis indicative of a predisposition of the tumor to undergo tumor invasionor tumor metastasis.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. In thefollowing, examples will be described to illustrate parts of theinvention. It is also understood that the terminology used herein is forthe purposes of describing particular embodiments.

EXPERIMENTAL Example 1—Affinities of Various AXL FC Constructs

FIG. 1 shows the four domains of AXL and the various combinations of AXLFc constructs made and tested.

The following AXL Fc constructs were made:

a. Full-length wild-type Fc fusion

b. Full-length AXL peptide 1 Fc fusion

c. AXL peptide 1 Fn(-) Fc fusion (this is the Fn- construct)

d. Full-length AXL peptide 1 Fc fusion with minor GAS6 binding siteknocked out

e. AXL peptide 1 Fn(-) Fc fusion, 3× gly4ser linker between Fc and AXL

f. AXL peptide 1 Fn(-) Fc fusion, 5× gly4ser linker between Fc and AXL

g. AXL peptide 1 A72V Fn(-) Fc fusion, 3× gly4ser linker between Fc andAXL

The following Table 1 outlines the affinities of the above constructs toGAS6, with wild-type AXL as a comparison.

TABLE 1 Construct Affinities AXL clone Fn domains Fc Linker K_(d) (pM)Wild-type Ig1 − None None 32.8 ± 0.63  AXL peptide 1 Ig1 − None None 2.7± 0.05 (a) Wild-type + hIgG None 9.2 ± 0.17 (b) AXL peptide 1 + hIgGNone 0.4 ± 0.01 (c) AXL peptide 1 − hIgG None 2.6 ± 0.05 (d) AXL peptide1 + hIgG None 2.6 ± 0.10 (−) minor site (e) AXL peptide 1 − hIgG 3xgly₄ser 1.2 ± 0.03 (SEQ ID NO: 10) (f) AXL peptide 1 − hIgG 5x gly₄ser1.2 ± 0.03 (SEQ ID NO: 10) (g) AXL peptide 1 − hIgG 3x gly₄ser 0.3 ±0.00 A72V (SEQ ID NO: 10)

There are several conclusions that can be drawn from the data set inTable 1 above.

Fc-fusion constructs provide enhancements in affinity over the monomericforms. For example: wild-type AXL Ig1 (monomeric) has a ˜33 pM affinity,whereas wild-type Fc fusion has an affinity of ˜9 pM and AXL peptide 1Ig1 (monomeric) has an affinity of ˜3 pM, whereas AXL peptide 1-Fcfusion has an affinity of ˜0.4 pM

Significant affinity improvements for AXL peptide 1 over wild-type AXL.In addition, AXL peptide 1 plus the A72V mutation has a furtherenhancement in affinity, construct (e) compared to construct (g), overwild-type AXL.

The mechanism of increasing the affinity in the Fc fusion comes frommultivalent binding to a single GAS6 molecule. Specifically, one arm ofthe fusion binds to the major AXL binding site while the other binds theminor. This conclusion was based on the following experimental data:

-   -   a. AXL peptide 1 Ig1 (monomer) has the same affinity as the AXL        peptide 1 Fn(-) Fc fusion, (c) in the table above. This suggests        that simply having two copies of AXL is insufficient for        providing affinity improvement.    -   b. Full-length AXL peptide 1 with the minor binding site removed        has the same affinity as the monomer and the Fn(-) fusion. This        shows that the minor binding site has a definitive role in the        affinity improvements.    -   c. The Fn(-) constructs are arranged such that the minor binding        site is inaccessible to the large GAS6 molecule. The addition of        linkers between AXL and the Fc provide additional flexibility        and space, and with that a two-fold improvement in affinity is        obtained. This further supports the idea that the minor binding        site is important.

Overall, Example 1 shows that Fc fusions of the AXL ECD can haveimproved affinity to GAS6 compared to wild-type AXL. While not beingbound by theory, one mechanism underlying this improved affinity issimultaneous binding of one arm of the construct to the major AXLbinding site on GAS6 and the other arm to the minor AXL binding site onGAS6.

Example 2—Affinities of Various AXL FC Constructs

Sequence: AXL peptide 2-Fc. The AXL peptide 2 includes amino acids 1-131of AXL, has the Ig2 domain deleted and has both the FN domains deleted.

This construct includes amino acids 1-131 of AXL fused to the humanIgG1, with a single Gly4Ser (SEQ ID NO:10) linker connecting the twodomains (see FIG. 2). Additionally, constructs can include variousmutations in the AXL portion of the molecule, as described by thepresent invention. For example, embodiments can include having AXLpeptide 1 Ig1 Fc fusion or AXL peptide 1 plus A72V.

The affinity of AXL peptide 1 Ig1-Fc to human GAS6 has been measured tobe 1.7 +/−0.03 pM.

Overall, Example 2 shows that Fc fusions of the AXL Ig1 domain can haveimproved affinity to GAS6 compared to wild-type AXL.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor only and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the appended claims.

LISTING OF SEQUENCES:SEQ ID NO: 1 is the amino acid sequence of human wild type AXLMAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSNDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGASEQ ID NO: 2 is the amino acid sequence of human wild type MERMGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFADDSSEGSEVLMSEQ ID NO: 3 is the amino acid sequence of human wild type Tyro3MALRRSMGRPGLPPLPLPPPPRLGLLLAALASLLLPESAAAGLKLMGAPVKLTVSQGQPVKLNCSVEGMEEPDIQWVKDGAVVQNLDQLYIPVSEQHWIGFLSLKSVERSDAGRYWCQVEDGGETEISQPVWLTVEGVPFFTVEPKDLAVPPNAPFQLSCEAVGPPEPVTIVWWRGTTKIGGPAPSPSVLNVTGVTQSTMFSCEAHNLKGLASSRTATVHLQALPAAPFNITVTKLSSSNASVAWMPGADGRALLQSCTVQVTQAPGGWEVLAVVVPVPPFTCLLRDLVPATNYSLRVRCANALGPSPYADWVPFQTKGLAPASAPQNLHAIRTDSGLILEWEEVIPEAPLEGPLGPYKLSWVQDNGTQDELTVEGTRANLTGWDPQKDLIVRVCVSNAVGCGPWSQPLVVSSHDRAGQQGPPHSRTSWVPVVLGVLTALVTAAALALILLRKRRKETRFGQAFDSVMARGEPAVHFRAARSFNRERPERIEATLDSLGISDELKEKLEDVLIPEQQFTLGRMLGKGEFGSVREAQLKQEDGSFVKVAVKMLKADIIASSDIEEFLREAACMKEFDHPHVAKLVGVSLRSRAKGRLPIPMVILPFMKHGDLHAFLLASRIGENPFNLPLQTLIRFMVDIACGMEYLSSRNFIHRDLAARNCMLAEDMTVCVADFGLSRKIYSGDYYRQGCASKLPVKWLALESLADNLYTVQSDVWAFGVTMWEIMTRGQTPYAGIENAEIYNYLIGGNRLKQPPECMEDVYDLMYQCWSADPKQRPSFTCLRMELENILGQLSVLSASQDPLYINIERAEEPTAGGSLELPGRDQPYSGAGDGSGMGAVGGTPSDCRYILTPGGLAEQPGQAEHQPESPLNETQRLLLLQQGLLPHSSCSEQ ID NO: 4 is the amino acid sequence region L295-T317 of human GAS6LRMFSGTPVIRLRFKRLQPTSEQ ID NO: 5 is the amino acid sequence region E356-P372 of human GAS6ElVGRVTSSGPSEQ ID NO: 6 is the amino acid sequence region R389-N396 of human GAS6RNLVIKVNSEQ ID NO: 7 is the amino acid sequence region D398-A406 of human GAS6DAVMKIAVASEQ ID NO: 8 is the amino acid sequence region E413-H429 of human GAS6ERGLYHLNLTVGIPFHSEQ ID NO: 9 is the amino acid sequence region W450-M468 of human GAS6WLNGEDTTIQETVVNRM SEQ ID NO: 10 is the (GLY)₄SER linker sequence GGGGS

1-80. (canceled)
 81. An inhibitor agent selected from the groupconsisting of (a) an inhibitor of AXL, MER or Tyro3-GAS6 interaction,(b) an inhibitor of AXL, MER and/or Tyro3 activity, and (c) an inhibitorof GAS6 activity, and wherein the inhibitor agent binds to GAS6 withincreased affinity compared to wild-type AXL, MER or Tyro3.
 82. Theinhibitor agent of claim 81 wherein the inhibitor agent binds to two ormore epitopes on a single GAS6, optionally where at least one of theepitopes is the major or minor AXL, MER or Tyro3 binding site of GAS6.83. The inhibitor agent of claim 82, wherein the inhibitor comprises asoluble AXL variant polypeptide, wherein said soluble AXL variantpolypeptide: lacks the AXL transmembrane domain; lacks both AXLfibronectin (FN) domains; has a set of amino acid substitutions relativeto SEQ ID NO: 1, selected from the group consisting of: 1) Ala72Val; 2)Asp87Gly; 3) Val92Ala, Val92Gly or Val92Asp; 4) Ala19Thr; 5) Thr23Met;6) Glu26Gly; 7) Glu27Gly or Glu27Lys; 8) Gly32Ser; 9) Asn33Ser; 10)Thr38lle; 11) Thr44Ala; 12) His61Tyr; 13) Asp65Asn; 14) Ser74Asn; 15)Gln78Glu; 16) Val79Met; 17) Gln86Arg; 18) Asp88Asn; 19) lle90Met orlle90Val; 20) lle97Arg; 21) Thr98Ala or Thr98Pro; 22) Thr105Met; 23)Gln109Arg; 24) Val112Ala; 25) Phe113Leu; 26) His116Arg; 27) Thr118Ala;28) Gly127Arg or Gly127Glu; 29) Glu129Lys; 30) Glu26Gly, Val79Met;Val92Ala and Gly127Glu; 31) Asp87Gly, Val92Ala and Gly127Arg; 32)Gly32Ser, Val92Ala and Gly127Arg; 33) Gly32Ser, Asp87Gly and Gly127Arg;34) Gly32Ser, Asp87Gly, and Val92Ala; and 35) Asp87Gly and Val92Ala; andwherein the inhibitor further comprises an Fc domain linked to thesoluble AXL variant polypeptide by a linker comprising from 1 to 5(GLY)4SER (SEQID NO: 10) units.
 84. A pharmaceutical formulationcomprising an inhibitor of claim 81 and a pharmaceutically acceptableexcipient.
 85. A method of reducing growth or metastasis of a tumor thatexpresses GAS6, the method comprising administering to a patient with atumor that expresses GAS6 an effective dose of the pharmaceuticalformulation of claim 84.